Quantitation of canine coronavirus RNA in the faeces of dogs by TaqMan RT-PCR.
Quantitation of canine coronavirus RNA in the faeces of dogs by TaqMan RT-PCR.
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DOI：
10.1016/j.jviromet.2004.03.012
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发表时间：
2004-08
影响因子：
3.1
通讯作者：
Buonavoglia C
中科院分区：
文献类型：
--
作者：
Decaro N;Pratelli A;Campolo M;Elia G;Martella V;Tempesta M;Buonavoglia C
建立了TaqMan®荧光逆转录聚合酶链反应（RT-PCR）方法，用于检测和定量自然或实验感染犬粪便中的犬冠状病毒（CCoV） RNA。针对ORF5 （M基因）的冠状病毒荧光RT-PCR检测比针对相同基因的传统RT-PCR检测更敏感，显示出10拷贝的冠状病毒标准RNA的检测限，并且在10到108拷贝之间呈线性，允许对具有广泛冠状病毒RNA负载的样品进行定量。采用常规RT-PCR和荧光RT-PCR同时检测了78份腹泻犬粪便样本：29份两种技术均为阴性，而27份常规RT-PCR检测呈阳性，48份采用已建立的冠状病毒荧光测定法检测呈阳性。一份常规RT-PCR阳性的样品在荧光分析中没有信号。此外，通过荧光测定法对实验感染犬粪便样本中的冠状病毒脱落情况进行了28天的监测。新型冠状病毒荧光RT-PCR检测具有灵敏度高、操作简单、重复性好、动态范围广、通量高的特点，特别适用于新型冠状病毒疫苗的疗效试验。
A TaqMan® fluorogenic reverse transcriptase-polymerase chain reaction (RT-PCR) assay was developed for the detection and quantitation of canine coronavirus (CCoV) RNA in the faeces of naturally or experimentally infected dogs. The CCoV fluorogenic RT-PCR assay, which targeted the ORF5 (M gene), was more sensitive than a conventional RT-PCR assay targeting the same gene, showing a detection limit of 10 copies of CCoV standard RNA, and was linear from 10 to 108 copies, allowing quantitation of samples with a wide range of CCoV RNA loads. A total of 78 faecal samples of diarrhoeic dogs were tested simultaneously by conventional and fluorogenic RT-PCR: 29 were negative by both techniques, whereas 27 tested positive by conventional RT-PCR and 48 by the established CCoV fluorogenic assay. One sample, which was positive by conventional RT-PCR, gave no signal in the fluorogenic assay. In addition, by the fluorogenic assay CCoV shedding in the faecal samples of an experimentally infected dog was monitored for 28 days. The high sensitivity, simplicity and reproducibility of the CCoV fluorogenic RT-PCR assay, combined with its wide dynamic range and high throughput, make this method especially suitable for efficacy trials on CCoV vaccines.
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影响因子：
3.1
作者：
Pratelli A;Martella V;Pistello M;Elia G;Decaro N;Buonavoglia D;Camero M;Tempesta M;Buonavoglia C
通讯作者：
Buonavoglia C
DOI：
10.1073/pnas.88.16.7276
发表时间：
1991-08-01
影响因子：
11.1
作者：
HOLLAND, PM;ABRAMSON, RD;GELFAND, DH
通讯作者：
GELFAND, DH
影响因子：
9.4
作者：
Spackman, E;Senne, DA;Suarez, DL
通讯作者：
Suarez, DL
影响因子：
7
作者：
Heid, CA;Stevens, J;Williams, PM
通讯作者：
Williams, PM
影响因子：
3.1
作者：
Pratelli, A;Elia, G;Buonavoglia, C
通讯作者：
Buonavoglia, C